A proof of the DBRF-MEGN method, an algorithm for deducing minimum equivalent gene networks

Background

We previously developed the DBRF-MEGN (difference-based regulation finding-minimum equivalent gene network) method, which deduces the most parsimonious signed directed graphs (SDGs) consistent with expression profiles of single-gene deletion mutants. However, until the present study, we have not presented the details of the method's algorithm or a proof of the algorithm.

Results

We describe in detail the algorithm of the DBRF-MEGN method and prove that the algorithm deduces all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.

Conclusions

The DBRF-MEGN method provides all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.     

Somatic embryogenesis of tissue cultures of Papaver somniferum and Papaver orientale and its relationship to alkaloid and lipid metabolism

Transfer from complete to 2,4-D free Gamborg's B5-medium efficiently induced somatic embryogenesis in Papaver tissue cultures (P. somniferum and P. orientale). Embryogenesis was preceded by a strong temporary accumulation of triacylglycerols. In both tissue cultures large amounts of sanguinarine type alkaloids were present, which disappeared during regeneration in the P. orientale cultures but persisted in the P. somniferum cultures. In the P. somniferum cultures protopine and morphine type alkaloids (morphine, codeine, thebaine) appeared about 45 days after exchanging the medium. Thebaine was the main alkaloid in the P. somniferum embryoids accumulating up to 0.2 % of dry weight.     

Inactivation of aphidicolin by plant cells

Plant cells are endowed with an aphidicolin inactivating activity. Data on cultured cells show that the rate of inactivation depends on the cell type, Daucus carota cells being the most effective among the other tested materials (Oryza sativa and Nicotiana plumbaginifolia). Also germinating seedling of Haplopappus gracilis and of Citrullus vulgaris inactivate aphidicolin. Inactivation, which may lead to unexpected results when a prolonged incubation with the drug is required, as in the case of the induction of synchrony of the cell cycle by aphidicolin, can be controlled by appropriately choosing the experimental conditions.     

Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

Two novel techniques improve division and colony formation from protoplasts:

1. Plating in agarose stimulates colony formation of protoplasts from a wide range of species. Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium.
2. Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (e.g. Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida).

The combination of ‘agarose plating’ and ‘bead culture’ dramatically improved plating efficiencies of protoplasts in all species tested.     

High production of caffeine and related enzyme activities in callus cultures of Coffea arabica L.

Callus tissue culture of Coffea arabica L. cv Hybrido de Timor prepared from apical portions of orthotropic branches produced 49 to 92 times as much caffeine per unit weight of tissue as did the original explant. Cell-free extracts made from 42 to 54-day-old callus cultures in which active biosynthesis was occurring exhibited N-methyl-N ⁹-nucleoside hydrolase and N-methyltransferase enzyme activities. Similar cell-free extracts exhibited selective biodegradative activity in forming urea from xanthine. Biosynthetic substrate specificities are similar to those of the enzyme obtained from green coffee fruit and tea leaves, suggesting that callus cultures of C. arabica form caffeine in the same way as the coffee fruit and tea leaves.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites

Background

High throughput techniques have generated a huge set of biological data, which are deposited in various databases. Efficient exploitation of these databases is often hampered by a lack of appropriate tools, which allow easy and reliable identification of genes that miss functional characterization but are correlated with specific biological conditions (e.g. organotypic expression).

Results

We have developed a simple algorithm (DGSA = Database-dependent Gene Selection and Analysis) to identify genes with unknown functions involved in organ development concentrating on the heart. Using our approach, we identified a large number of yet uncharacterized genes, which are expressed during heart development. An initial functional characterization of genes by loss-of-function analysis employing morpholino injections into zebrafish embryos disclosed severe developmental defects indicating a decisive function of selected genes for developmental processes.

Conclusion

We conclude that DGSA is a versatile tool for database mining allowing efficient selection of uncharacterized genes for functional analysis.     

Absence of carious lesions at margins of glass-ionomer cement and amalgam restorations: An update of systematic review evidence

Background

A recent analysis of protein sequences deposited in the NCBI RefSeq database indicates that ~8.5 million protein sequences are encoded in prokaryotic and eukaryotic genomes, where ~30% are explicitly annotated as "hypothetical" or "uncharacterized" protein. Our Comparison of Protein Active-Site Structures (CPASS v.2) database and software compares the sequence and structural characteristics of experimentally determined ligand binding sites to infer a functional relationship in the absence of global sequence or structure similarity. CPASS is an important component of our Functional Annotation Screening Technology by NMR (FAST-NMR) protocol and has been successfully applied to aid the annotation of a number of proteins of unknown function.

Findings

We report a major upgrade to our CPASS software and database that significantly improves its broad utility. CPASS v.2 is designed with a layered architecture to increase flexibility and portability that also enables job distribution over the Open Science Grid (OSG) to increase speed. Similarly, the CPASS interface was enhanced to provide more user flexibility in submitting a CPASS query. CPASS v.2 now allows for both automatic and manual definition of ligand-binding sites and permits pair-wise, one versus all, one versus list, or list versus list comparisons. Solvent accessible surface area, ligand root-mean square difference, and Cβ distances have been incorporated into the CPASS similarity function to improve the quality of the results. The CPASS database has also been updated.

Conclusions

CPASS v.2 is more than an order of magnitude faster than the original implementation, and allows for multiple simultaneous job submissions. Similarly, the CPASS database of ligand-defined binding sites has increased in size by ~ 38%, dramatically increasing the likelihood of a positive search result. The modification to the CPASS similarity function is effective in reducing CPASS similarity scores for false positives by ~30%, while leaving true positives unaffected. Importantly, receiver operating characteristics (ROC) curves demonstrate the high correlation between CPASS similarity scores and an accurate functional assignment. As indicated by distribution curves, scores ≥ 30% infer a functional similarity. Software URL: http://cpass.unl.edu.     

6-OHDA generated ROS induces DNA damage and p53- and PUMA-dependent cell death

Background

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons in the substantia nigra (SN), resulting in tremor, rigidity, and bradykinesia. Although the etiology is unknown, insight into the disease process comes from the dopamine (DA) derivative, 6-hydroxydopamine (6-OHDA), which produces PD-like symptoms. Studies show that 6-OHDA activates stress pathways, such as the unfolded protein response (UPR), triggers mitochondrial release of cytochrome-c, and activates caspases, such as caspase-3. Because the BH3-only protein, Puma (p53-upregulated mediator of apoptosis), is activated in response to UPR, it is thought to be a link between cell stress and apoptosis.

Results

To test the hypothesis that Puma serves such a role in 6-OHDA-mediated cell death, we compared the response of dopaminergic neurons from wild-type and Puma-null mice to 6-OHDA. Results indicate that Puma is required for 6-OHDA-induced cell death, in primary dissociated midbrain cultures as well as in vivo. In these cultures, 6-OHDA-induced DNA damage and p53 were required for 6-OHDA-induced cell death. In contrast, while 6-OHDA led to upregulation of UPR markers, loss of ATF3 did not protect against 6-OHDA.

Conclusions

Together, our results indicate that 6-OHDA-induced upregulation of Puma and cell death are independent of UPR. Instead, p53 and DNA damage repair pathways mediate 6-OHDA-induced toxicity.     

Osteopathic manipulative treatment and its relationship to autonomic nervous system activity as demonstrated by heart rate variability: a repeated measures study

Background

Osteopathic manipulative treatment (OMT) and ultrasound physical therapy (UPT) are commonly used for chronic low back pain. Although there is evidence from a systematic review and meta-analysis that OMT generally reduces low back pain, there are no large clinical trials that specifically assess OMT efficacy in chronic low back pain. Similarly, there is a lack of evidence involving UPT for chronic low back pain.

Methods

The OSTEOPAThic Health outcomes In Chronic low back pain (OSTEOPATHIC) Trial is a Phase III randomized controlled trial that seeks to study 488 subjects between August 2006 and June 2010. It uses a 2 × 2 factorial design to independently assess the efficacy of OMT and UPT for chronic low back pain. The primary outcome is a visual analogue scale score for pain. Secondary outcomes include back-specific functioning, generic health, work disability, and satisfaction with back care.

Conclusion

This randomized controlled trial will potentially be the largest involving OMT. It will provide long awaited data on the efficacy of OMT and UPT for chronic low back pain.

Trial registration

http://www.clinicaltrials.gov, NCT00315120     

A special issue on DNA damage response and genome stability

A novel, cancer-fighting function was recently discovered for Smad ubiquitination regulatory factor 2 (Smurf2).     

Multiscale Hy3S: Hybrid stochastic simulation for supercomputers

Background

DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell.

Results

We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100, 000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods.

Conclusion

Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.     

Roles of glycosaminoglycans and glycanmimetics in tumor progression and metastasis

Various tumor cells exhibit structural alterations in the sulfated modifications to glycosaminoglycans (GAGs). The altered expression of chondroitin sulfate (CS) and heparan sulfate (HS) on the surfaces of tumor cells is known to play a key role in malignant transformation and tumor metastasis. The receptor molecule for the CS chains containing E-disaccharide units (CS-E) expressed on Lewis lung carcinoma (LLC) cells was recently revealed to be Receptor for Advanced Glycation End-products (RAGE). RAGE is also involved in the development of various pathological conditions including aging, infection, pulmonary fibrosis, diabetes, and Alzheimer’s disease, by binding to a wide range of ligands. RAGE binds strongly not only to CS-E, but also to HS-expressing LLC cells. Recombinant RAGE bound CS-E and HS with high affinity. Furthermore, in a mouse model, the colonization of the lungs by LLC cells was inhibited by intravenously injected CS-E, an anti-CS-E antibody, or an anti-RAGE antibody. These findings demonstrated that RAGE is at least one of the critical receptors for CS and HS chains expressed on the tumor cell surface and is involved in experimental lung metastasis, and also that CS/HS and RAGE are potential molecular targets for the treatment of pulmonary metastasis. We, hence, reviewed these findings and also several chemically synthesized small GAGmimetics that exhibit potent anti-metastatic and/or anti-tumor activities.     

siMBa—a simple graphical user interface for the Bayesian phylogenetic inference program MrBayes

MrBayes is a program that uses a Bayesian framework for inferring phylogenetic relationships. As MrBayes is a command-line-driven program, users acquainted to programs with graphical user interfaces will not find it easy to operate, especially as it requires a complex input format for the data to be analysed. We thus developed siMBa (simple MrBayes), a simple graphical user interface for MrBayes. This tool gives the user interactive control over most of the parameters and also facilitates the input of a multiple sequence alignment, as any widely used format can be used. siMBa is coded in Perl using the Tk module. Executables are provided for Windows, Linux, and Macintosh. The Perl codes, along with executables for different operating system, are freely available to download from [http://www.thines-lab.senckenberg.de/simba].     

Genome skimming identifies polymorphism in tern populations and species

Background

Large-scale protein structure alignment, an indispensable tool to structural bioinformatics, poses a tremendous challenge on computational resources. To ensure structure alignment accuracy and efficiency, efforts have been made to parallelize traditional alignment algorithms in grid environments. However, these solutions are costly and of limited accessibility. Others trade alignment quality for speedup by using high-level characteristics of structure fragments for structure comparisons.

Findings

We present ppsAlign, a parallel protein structure Alignment framework designed and optimized to exploit the parallelism of Graphics Processing Units (GPUs). As a general-purpose GPU platform, ppsAlign could take many concurrent methods, such as TM-align and Fr-TM-align, into the parallelized algorithm design. We evaluated ppsAlign on an NVIDIA Tesla C2050 GPU card, and compared it with existing software solutions running on an AMD dual-core CPU. We observed a 36-fold speedup over TM-align, a 65-fold speedup over Fr-TM-align, and a 40-fold speedup over MAMMOTH.

Conclusions

ppsAlign is a high-performance protein structure alignment tool designed to tackle the computational complexity issues from protein structural data. The solution presented in this paper allows large-scale structure comparisons to be performed using massive parallel computing power of GPU.     

Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D

Background

Patients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.

Methods

Lung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES (Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.

Results

TNF-α increased the expression of RANTES (307.84 ± 33.56 versus 207.64 ± 31.27 pg/ml of control, p = 0.025) and eotaxin (108.97 ± 10.87 versus 54.00 ± 5.29 pg/ml of control, p = 0.041). Melatonin(10^-10 to 10^-6M) alone didn't change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES (410.88 ± 52.03, 483.60 ± 55.37, 559.92 ± 75.70, 688.42 ± 95.32, 766.39 ± 101.53 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7,10^-6M melatonin, respectively) and eotaxin (151.95 ± 13.88, 238.79 ± 16.81, 361.62 ± 36.91, 393.66 ± 44.89, 494.34 ± 100.95 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7, 10^-6M melatonin, respectively) expression in a dose dependent manner in A549 cells (compared with TNF-α alone, P < 0.05). The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.

Conclusion

Taken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell.     

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

Background

Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved.

Results

Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya (S. c. hoya) and Pycnonotus sinensis (P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished.

Conclusion

In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.